Project Summary/Abstract Cancer remains the second most common cause of death in the US. Despite tremendous progress in the understanding of cancer biology, translation into clinical applications has been slow. Gene therapy could be one of the most direct ways to translate knowledge about genetic aberrations in cancer into effective therapy. It has been demonstrated that restoration of a single tumor suppressor gene (p53) in vivo results in tumor regression and many excellent targets for gene therapy are known. However, current gene therapy vector systems provide very inefficient gene transfer. This problem has been difficult to overcome for many years, limiting clinical efficacy. The main objective of this proposal is to circumvent the obstacle of inefficient gene transfer through the induction of a strong bystander effect using protein transduction technology. In addition, we hypothesize that targeting the tumor stroma is a critical aspect for an effective gene therapy strategy. There is strong evidence that the tumor stroma plays an important role in tumor development, progression, and metastasis, a fact that is not addressed with most gene therapy vector systems. Hence, the second objective of the proposal is to target the tumor stroma. The central hypothesis of the proposed work is that a vector-expressed secreted protein that is fused to a cell penetrating peptide (CPP) can induce a bystander effect and target the tumor stroma. p53 will be studied as the effector protein, because of its non-toxic effects on normal cells and established safety profile with conventional gene therapy vectors. Of particular interest for this proposal are the recently discovered functions of p53 in stromal cells that contribute to its tumor suppressor effect. Carcinoma-associated-fibroblasts (CAF) communicate with cancer cells through the secretion of cytokines, such as SDF-1, VEGF or IL-6, and support tumor growth and metastasis in a paracrine fashion. Expression of several of these cytokines is down regulated by p53. In addition, p53 alterations in tumor-associated stromal cells discordant from the epithelial compartment have been found at a high frequency in clinical samples, and stromal p53 ablation in animal experiments leads to reduced tumor latency and rapid tumor progression. These findings support the hypothesis that p53 transfer into CAF will suppress the tumor supportive function of these cells by altering their cytokine profile, or induction of cell death in the presence of p53 aberration. The proposed experiments will test whether the vector system can induce a bystander effect, whether CAF can be targeted, and whether the tumor-growth and metastasis supportive functions of these cells can be reversed. The presented approach to induce a bystander effect and to target the tumor stroma is novel, and if successful, could have numerous applications. In addition, the proposed work will contribute to the understanding of the importance of p53 in the tumor stroma.